枯草芽孢杆菌酸性内切葡聚糖酶的分离纯化及酶学性质

Purification and characterization of acid cellulase fromBacillussubtilis

将来源于藏猪粪便的枯草芽孢杆菌BY-4菌株接种于含4%麸皮的产酶培养基中,37℃、220r/min培养48h,发酵上清液经HiTrap Phenyl HP疏水作用层析、HiPrep 16/60Sephacryl S-100凝胶过滤层析分离纯化得到一个内切葡聚糖酶组分,纯化倍数为15.5,回收率为25.0%。经SDS-PAGE、酶谱分析鉴定,两步分离纯化后为电泳纯的纤维素酶,相对分子量约为55 000。酶学性质研究表明,该酶的最适反应pH为4.5,最适反应温度为60℃。Mg2+、Zn2+对该酶有一定的激活作用,而Mn2+、Co2+、Ag+对该酶产生了抑制作用。

Bacillus subtilis BY-4,isolated from Tibetan pig,had been cultured in the fermentation medium(containing 4% wheat bran)at 37 ℃ with agitation speed of 220r/min for 48 h.The crude enzyme was purified by hydrophobic interaction chromatography on HiTrap Phenyl HP column and gel filtration chromatography on HiPrep 16/60 Sephacryl S-100 column.A purified endoglucanase was achieved with purification fold of 15.5and a recovery rate of 25.0%.SDS-PAGE and zymogram assay revealed an electrophoretically pure cellulase with apparent molecular weights of 55 000.The optimal values of pH and temperature on purified cellulase response were found to be 4.5and 60℃,respectively.The cellulase can be activated by Mg2+and Zn2+,while inhibited by Mn2+,Co2+and Ag+.